The pellet was resuspended in the buffer and recentrifuged at 12,000g for 15 minutes; the resulting pellet was resuspended in a minimal volume of the buffer and constituted the mitochondria enriched fraction. Cells or cellular fractions were lysed in 20 mM HEPES, pH 7.2, 150 mM NaCl, CHIR-99021 chemical structure 1 mM ethylene glycol tetraacetic acid, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 2 mM sodium phosphate, and protease inhibitor cocktail, followed by 10% SDS-PAGE and western blotting using monoclonal antibodies 5B-3B1 against HCV NS5B,19, 23 C7-50 against HCV core,19, 23 rabbit anti-cytochrome c (1:400, Abcam), anti-cleaved caspase 3 (1:1000, Cell Signaling Technology), anti-β-subunit

of CV (MitoSciences) or anti-β-actin

(Sigma) as primary antibodies. A two-tailed Student t test was applied to evaluate the statistical significance of differences measured from the datasets reported. P < 0.05 was considered statistically significant. We have shown that HCV protein expression causes a dose- and time-dependent alteration of the main bioenergetic functions of mitochondria19, 20 with the major dysfunctions observed already 36-48 hours after HCV protein induction.19 To assess a possible involvement of the MPTP in HCV protein-mediated mitochondrial deregulation, we LDE225 molecular weight tested the effect of alisporivir, a non-immunosuppressive analog of the MPTP inhibitor CsA. Opening of the MPTP causes collapse of the respiration-driven mtΔΨ.12 As shown previously19 and confirmed in Fig. 1A, HCV protein expression for 48 hours resulted in a marked reduction of the mtΔΨ, as assessed by the fluorescent probe TMRE. Alisporivir prevented the dissipation of the mtΔΨ in a dose-dependent manner. HCV protein-induced 上海皓元医药股份有限公司 collapse of the mtΔΨ was completely prevented at 0.125 μM, a concentration that has been shown to significantly reduce

HCV RNA levels in the HCV replicon system4 (Fig. 1A,B). No significant effects were observed in noninduced control cells treated by low concentrations of alisporivir, whereas a slight hyperpolarization was detected at the highest concentration. Importantly, treatment with 0.125 μM alisporivir did not affect the expression level of HCV proteins after 48 hours of induction (Fig. 1C). Opening of the MPTP may cause leakage of low molecular weight mitochondrial respiratory substrates.24 To test this possibility, we performed measurements of respiratory rates in intact cells by high-resolution oxymetry. As shown in Fig. 2, HCV protein expression for 48 hours resulted in a significant inhibition of the cyanide-sensitive dioxygen consumption under endogenous respiratory setting both in the absence and in the presence of either oligomycin (an ATP-synthase inhibitor) or FCCP (an oxidative phosphorylation uncoupler).